Methods of single step determination of the mass of nucleic acids in the mass spectrometer have been developed mainly for sequencing (H. Koster et al., Nature Biotechnology 14, 1123-1128, 1996). There are, however, a number of problems with the direct analysis of DNA in a mass spectrometer at present. One is fragmentation of the DNA. The longer a molecule to be analysed is, the greater the degree of fragmentation. This gives rise to mass spectra that are very difficult to interpret. However improvements are envisaged, using modified nucleotide analogues that are resistant to fragmentation within a mass spectrometer.
A further problem of great significance is accurate mass measurement of moderately large biomolecules. This resolution problem limits read lengths of DNA sequences achievable to a significant degree. At present the absolute limit on direct mass analysis of Sanger ladders is determination of sequences of about 100 bases in length and is nearer 30 to 40 bases for practical purposes.
GB 9719284.3 describes the use of nucleic acid hybridisation probes cleavably linked to mass labels for the analysis of nucleic acids. GB 9719284.3 describes a method of sequencing nucleic acids exploiting mass labelled sequencing primers or nucleotides to generate Sanger ladders. This sequencing method uses capillary electrophoresis mass spectrometry as the mass spectrometry method to analyse the mass labelled Sanger ladders generated. These methods require a two-stage analysis; a sizing step which determines the lengths of each nucleic acid in a population, i.e. the number of nucleotides that comprise its linear sequence, followed by identification of the mass label each nucleic acid carries.